In measurement, evaluation or control of adhesion of cells to biological substrate material in culture solution and the subsequent biological reaction (spreading, differentiation and the like), hydration phenomena of biomolecules, living tissues, biological substrate material and the like is significant. The hydration structure shows a three-dimensional structure formed with (1) interaction between sample surface and water molecules and (2) interaction including hydrogen bond between water molecules, in the interface between the sample and culture solution with water as a main component. It is considered that so-called biocompatibility typified by adhesion between an inner wall of an artificial blood vessel and red blood cells or the like is closely related to the hydration structure. Further, the unevenness of the sample surface in the culture solution, the electric potential distribution, the composition distribution and the array structure of the molecules or protein and the like relate to the biological reaction of the biomolecules, the living tissues, the biological substrate material and the like in the culture solution, and they are specially significant characteristics.
Conventionally, linear or nonlinear optical microscopes based on Raman spectroscopy, second harmonic generation spectroscopy (hereinafter referred to as SHG), sum frequency generation (hereinafter referred to as SFG) spectroscopy, and the like, are used for observation or measurement of the interface between the sample such as biomolecules, living tissues, biological substrate material or the like in the culture solution, and the culture solution. The SFG spectroscopy measures the intensity of scattered light from a region without the inversion symmetry in a sample by nonlinear optical phenomenon between scattering of infrared incident light (Raman scattering) related to molecular vibration of molecules included in the sample and visible incident light. Especially it is possible to measure the ordered structure of water molecules related to the hydration structure in the interface between the sample and the culture solution. As a nonlinear optical microscope, microscopes using a nonlinear optical method to surface-selectively observe the interaction between a probe and a target with e.g. SHG or SFG originated from water molecules, solvent molecules, or a marker in the vicinity of the interface, are known. However, the lateral resolution in these optical microscopes is higher than 100 nm or typically about 1 μm.
On the other hand, scanning probe microscopes have atomic force microscopy (hereinafter referred to as AFM) as a basis. As an example of the scanning probe microscope, Kelvin probe force microscope is known. The Kelvin probe force microscope is based on a method of mapping electrostatic force distribution by scanning a conductive probe on the sample surface while detecting an electrostatic force acting between a cantilever having the conductive probe and the sample as deflection of the cantilever. As the atomic force and the like other than the electrostatic force are also applied to the conductive probe, it is necessary to separate the electrostatic force from the other interaction. For this purpose, first, the cantilever is vibrated, and the distance between the conductive probe and the sample is adjusted so as to keep a vibrational amplitude, which reduces due to the atomic force to act upon contact between the conductive probe and the sample, constant. With this configuration, the position of the probe in a height direction of the sample surface is determined, and in a state where the conductive probe is away from the sample surface by a predetermined distance, an electrostatic force as a long-range force is detected from phase change of the vibration of the cantilever.
In the scanning probe microscope, generally, lateral resolution of about 1 nm by unevenness measurement is expected, and lateral resolution of about 10 nm by electrostatic force and optical measurement is expected. However, as an interaction region between the probe and the sample is limited to about a diameter of the tip of the probe, it is generally difficult to particularly realize a scanning probe microscope to use a weak signal physical quantity like the nonlinear optical method.
To realize the Raman spectroscopy with a scanning probe microscope by compensating the weak signal, various techniques related to tip-enhanced Raman detection method using surface-enhanced Raman scattering with a probe have been proposed. As one of these techniques, for example, a probe microscope, in which the depth of liquid held on a sample table is smaller than the length of a probe attached to the tip of an oscillator by using a holder cover 11 and a spacer 15 provided on a sample surface, to suppress reduction of a quality factor due to viscous resistance, has been proposed (Patent Literature 1). Further, a probe microscope which periodically displaces the probe position of the oscillator and controls the relative distance between the probe and the sample table, for synchronization with irradiation of pulsed laser light, to maximize tip-enhanced detection efficiency, has been proposed (Patent Literature 2).